The present invention relates generally to N-acetylmuramidase M1 and more particularly to microbial N-acetylmuramidase M1, to DNA sequences encoding N-acetylmuramidase M1, to the polypeptide products of recombinant expression of these DNA sequences, to peptides whose sequences are based upon the amino acid sequences deduced from these DNA sequences, to antibodies specific for such proteins and peptides, to procedures for the detection and quantitation of such proteins and nucleic acids related thereto, as well as to procedures relating to the development of therapeutic agents utilizing N-acetylmuramidase M1.
The occurrence of bacteriolytic enzymes in animals, plants, and microorganisms is widespread. These enzymes are categorized into three classes based on their mechanism of action on the carbohydrate polymers, such as peptidoglycans, comprising bacterial cell walls. One class of enzyme, the glycosidases, degrades cell walls by acting on the linear sequence of N-acetyl-D-glucosamine and N-acetyl muramic acid residues. A second class, the endopeptidases, splits bonds within peptides and cross linkages between peptides. A third class, the amidases, hydrolyzes linkages between the glycan and peptide moleties.
Those glycosidases that hydrolyze the .beta.-1,4-glycosidic bonds in the polysaccharide backbone of peptidoglycans are known as lysozymes (.beta.-1,4-N-acetylmuramidases). Lysozymes from a variety of sources, including animal, plant, and microbial, are classified into four distinct types on the basis of the homology of their amino acid sequence: i) chicken, ii) phage, iii) goose, and iv) fungal (Chalaropsis). see, Jolles, et al., Molec. Cell. Biochem., 63:165-189 (1984). There is no obvious sequence homology between one class of lysozymes and another class although X-ray structure analyses have shown that the three-dimensional structures of the first three types are similar to one another.
The bacterium Streptomyces globisporus produces two kinds of lysozyme, M1 and M2. Using the culture liltrate of S. globisporus, Yokogawa, K., et al., in Antimicrob. Ag. Chemother., 6:156 (1974), and Yokagawa, et al., in Agr. Biol. Chem., 39:1533 (1975), described the purification of mutanolysin, which contains multiple enzymatic activities including M1 and M2. Both M1 and M2 were found to be N-acetylmuramidases. However, the M1 enzyme (MW-20,000) was noted to show a greater lyric specificity towards Streptococcus mutans cell walls than the M2 enzyme (MW11,000). M1 and M2 also differ from each other in amino acid composition, immunological properties, and modes of lytic action. Kawata, S., et al., Agric. Biol. Chem., 47:1501 (1983). The hydrolyzing action of M1 (composed of 186 amino acid residues) is independent of the presence of 0-acetyl groups on muramic acid residues in the peptidoglycan moiety, while the action of M2 (composed of 99 amino acid residues) is suppressed by the presence of such groups. Thus, M1 is more similar to the Chalaropsis type lysozyme class in that both enzymes have N,O-diacetylmuramidase activity. M2 is more similar to the chicken type lysozyme class in that both enzymes cannot efficiently lyse O-acetylated peptidoglycan. Neither M1 nor M2 has been sequenced nor has the gene for either enzyme been isolated. Only preliminary X-ray crystallographic information is available and only for the M1 lysozyme. Harada, et al., J. Mol. Biol., 207:851-852 (1989). The only described attempts to isolate the gene for a B-1,4-Nacetylmuramidase from Streptomyces has been by Birr, E., et al., Appl. Microbiol. Biotechnol., 30:358 (1989). Lysozyme deficient mutants of Streptomyces coelicolor "Muller" were generated and then transformed with wild type S. coelicolor "Muller" genomic DNA. A 2.9 kb insert was identified and shown to restore lysozyme production in the mutants. However, there was no evidence demonstrating whether the 2.9 kb insert contained a structural gene for lysozyme.
N-acetylmuramidase M1 has a broad spectrum bacteriolytic activity and is particularly efficient in lysing lysozyme-resistant bacteria, such as Streptococcus and Lactobacillus. Evidence implicating involvement of lysozyme-resistant peptidoglycans in the induction of inflammatory arthritis suggests that an agent capable of lysing such resistant peptidoglycans, such as N-acetylmuramidase M1, can be an effective agent against arthritis. Bacterial cell wall peptidoglycans (particularly those which are lysozyme-resistant) are potent stimulators of inflammatory and immunologic processes. For example, when the lysozyme-resistant peptidoglycan-polysaccharide complex (PG-PS) from group A Streptococci or Lactobacillus casei is injected into experimental animals, an inflammatory arthritis develops which closely parallels the syndrome observed in humans. Cromartie, et al., J. Exp. Med., 146:1585 (1977) and Lehman, et al., Arthritis Rheum., 26:1259 (1983). The severity of the disease appears to be directly proportional to the dose of PG-PS that is injected. It has also been demonstrated that rats which are injected with lysozyme-resistant O-acetylated peptidoglycans from Neisseria gonorrhea develop a severe arthritis. However, if the experiment is repeated using lysozyme-sensitive 0-acetylation-deficient peptidoglycan from Neisseria gonorrhea, there is a significant reduction in the inflammatory response. Fleming, et al., Infect. Immun., 52:600-608 (1986).
More recently, N-acetylmuramidase M1 has been shown to be effective in treating the arthritis that is caused by injecting group A Streptococcal PG-PS into rats. This arthritis manifests itself as an acute joint inflammation followed by a chronic recurrent erosive arthritis. N-acetylmuramidase M1 injected up to 3 days (approximately the time for peak acute inflammation) after the injection of group A streptococcal PG-PS results in a complete resolution of the acute arthritis, as well as the prevention of chronic joint disease. Furthermore, when the injection of N-acetylmuramidase M1 is delayed until 14 days (approximately the time when the chronic phase of arthritis begins) after the injection of PG-PS the severity of chronic inflammation still can be significantly reduced. Janusz, et al., J. Exp. Med., 160:1360-1374 (1984).
The ability of N-acetylmuramidase M1 to be used in the treatment of human arthritis depends upon whether lysozyme-resistant peptidoglycans play a role in initiating the human arthritic condition. It is known that numerous bacteria colonize the human gastrointestinal tract. During the life cycle of these bacteria, numerous cell wall components are generated which may localize in joint and synovial tissues. The arthropathic potential of microbial components in humans is apparent in that many gastrointestinal, genito-urinary, and skin infections have an associated inflammatory arthritis.
N-acetylmuramidase M1 has also been shown to be effective in lysing many strains of cariogenic bacteria which induce dental plaque and caries. Yokogawa, et al., Agr. Biol. Chem., 39:1533-1543 (1975). Thus, N-acetylmuramidase M1 could be incorporated into chewing gum, toothpaste or mouthwash for the treatment and prevention of dental caries. While a similar idea was proposed by Yokogawa, et al., Agr. Biol. Chem., 36:2055-2065 (1972) and Yoshimura, et al., U.S. Pat. No. 3,929,579 (1975), these investigators proposed that mutanolysin (a mixture of bacteriolytic enzymes from S. globisporus, including N-acetylmuramidase M1) would be used as the preventative agent against tooth decay.
The use of N-acetylmuramidase M1 to attack cariogenic bacteria would appear to have significant advantages over using traditional oral administration of antibiotics to eliminate these bacteria. One advantage is that once ingested, N-acetylmuramidase M1 is degraded by enzymes in the stomach; the enzyme does not circulate systemically the way an antibiotic does. Another advantage of using N-acetylmuramidase M1 is that the enzyme has a bacteriocidal activity, rather than the bacteriostatic activity found with certain antibiotics. Furthermore, many cariogenic bacteria have developed resistances to antibiotics, whereas there have not been any reported instances of cariogenic bacteria that are resistant to the action of N-acetylmuramidase M1.
N-acetylmuramidase M1 could be used in other pharmaceutical or industrial applications where it is beneficial to lyse bacteria that are sensitive to this enzyme. Thus, throat lozenges could contain N-acetylmuramidase M1 to prevent and treat throat infections. N-acetylmuramidase M1 could also be formulated into ointments or creams to combat skin infections. Finally, N-acetylmuramidase M1 could be used as a preservative for foods, pharmaceuticals, cosmetics or any other products susceptible to microbial decay. Currently most products are preserved using chemicals. N-acetylmuramidase M1 would have the advantage of serving as a natural preservative that is not toxic to humans or to the environment.
Clearly, because of its broader spectrum bacteriolytic activity, N-acetylmuramidase M1 has multiple uses as a therapeutic agent requiring bacteriolytic activity. However, to date no one has developed methods for producing isolated and purified N-acetylmuramidase M1 in substantial quantities via recombinant DNA technology. Thus, there continues to exist a need in the art for such methods. To date no one has provided the amino acid sequence for N-acetyl muramidase M1, nor the DNA sequence encoding the protein. The availability of DNA sequences encoding Nacetylmuramidase M1 would make possible the application of recombinant methods to the large-scale production of this protein in procaryotic and/or eucaryotic host cells, as well as DNA-DNA, DNA-RNA, and RNA-RNA hybridization procedures for the detection, quantification and/or isolation of nucleic acids associated with this protein. Possession of the protein, and/or knowledge of the amino acid sequence of this protein, would make possible, in turn, the development of monoclonal and polyclonal antibodies thereto (including antibodies to protein fragments or synthetic peptides modelled thereon) for the use in immunological methods for the detection and quantification of the protein as well as homologous proteins, in samples, as well as allowing the development of procedures relating to the development of therapeutic agents utilizing N-acetylmuramidase M1. Knowledge of the amino acid sequence of N-acetylmuramidase M1 would also make possible the application of techniques described as protein engineering, whereby the properties of the enzyme may be altered by changing DNA codons for specific amino acids of the enzyme. Thus, the stability, activity, effective pH range, temperature range, and the like of the enzyme may be altered to impart new and improved properties.